Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854;
Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854.
Proc Natl Acad Sci U S A. 2019 Apr 9;116(15):7308-7313. doi: 10.1073/pnas.1814170116. Epub 2019 Mar 22.
The initiation phase of HIV reverse transcription has features that are distinct from its elongation phase. The first structure of a reverse transcription initiation complex (RTIC) that trapped the complex after incorporation of one ddCMP nucleotide was published recently [Larsen KP, et al. (2018) 557:118-122]. Here we report a crystal structure of a catalytically active HIV-1 RT/dsRNA complex that mimics the state of the RTIC before the first nucleotide incorporation. The structure reveals that the dsRNA-bound conformation of RT is closer to that of RT bound to a nonnucleoside RT inhibitor (NNRTI) and dsDNA; a hyperextended thumb conformation helps to accommodate the relatively wide dsRNA duplex. The RNA primer 3' end is positioned 5 Å away from the polymerase site; however, unlike in an NNRTI-bound state in which structural elements of RT restrict the movement of the primer, the primer terminus of dsRNA is not blocked from reaching the active site of RT. The observed structural changes and energetic cost of bringing the primer 3' end to the priming site are hypothesized to explain the slower nucleotide incorporation rate of the RTIC. An unusual crystal lattice interaction of dsRNA with its symmetry mate is reminiscent of the RNA architecture within the extended vRNA-tRNA in the RTIC. This RT/dsRNA complex captures the key structural characteristics and components of the RTIC, including the RT conformational changes and interactions with the dsRNA primer-binding site region, and these features have implications for better understanding of RT initiation.
HIV 逆转录的起始阶段具有不同于延伸阶段的特征。最近发表了第一个捕获了一个 ddCMP 核苷酸掺入后的逆转录起始复合物（RTIC）的 RTIC 结构[Larsen KP，等（2018）557：118-122]。在这里，我们报告了一个催化活性 HIV-1 RT/dsRNA 复合物的晶体结构，该结构模拟了第一个核苷酸掺入前 RTIC 的状态。该结构揭示了 dsRNA 结合的 RT 构象更接近与非核苷 RT 抑制剂（NNRTI）和 dsDNA 结合的 RT 构象；超伸展的拇指构象有助于容纳相对较宽的 dsRNA 双链。RNA 引物 3' 端距离聚合酶位点 5 Å；然而，与 NNRTI 结合状态不同，在 NNRTI 结合状态下，RT 的结构元件限制了引物的运动，dsRNA 的引物末端不会被阻止到达 RT 的活性位点。引物 3' 端到达引发位点的观察到的结构变化和能量成本被假设解释了 RTIC 中核苷酸掺入率较慢的原因。dsRNA 与其对称伴侣之间的不寻常晶体格子相互作用让人想起 RTIC 中扩展 vRNA-tRNA 内的 RNA 结构。该 RT/dsRNA 复合物捕获了 RTIC 的关键结构特征和组成部分，包括 RT 的构象变化以及与 dsRNA 引物结合位点区域的相互作用，这些特征对更好地理解 RT 起始具有重要意义。
